(a) Schematic representation of IL-6 receptor assembly kinetics elicited by IL-6 wildtype (top panel), HyIL-6 (middle panel) and IL-6 mutants molecules (bottom panel). (b) Crystal structure of IL-6, in green, bound to gp130 and IL-6Rα ectodomains, in blue and pink respectively. Inlet highlights the IL-6/gp130 site-2 binding interface. Amino acids included in the library design are colored in red. (c) Schematic representation of IL-6 display in the yeast surface via aga2p-aga1p interaction. (d) Work-flow of IL-6 library selection process. Five rounds of selection were undertaken, starting with 1 μM of gp130 ectodomain and finishing with 1 nM. (e) Representative gp130 staining of the selected IL-6 library. The five rounds of selections were incubated with 1 μM of biotinylated gp130 for 1 hr followed by 15 min incubation with SA-alexa647. Early rounds exhibit weak binding to gp130, but as the library converged into few high affinity clones, the gp130 staining improve significantly. (f) Dose/Response gp130 binding curves performed in single yeast colonies, each encoding a different IL-6 variant. Gp130 concentration started at 1 μM, and eight different concentrations in a 1/3 dilution series were tested.